The integrated Abelson murine leukemia virus (A-MuLV) genome cloned in bacteriophage vector was used to localize viral genetic sequences required for transformation. Comparison of the biological activity of cloned A-MuLV genomic and subgenomic fragments revealed that subgenomic clones which lacked 5' LTR and adjoining sequences were not biologically active. In contrast, subgenomic clones which lacked 3' LTR and as much as 1.3 kbp of the A-MuLV-specific abl gene, were as efficient as wild-type virus DNA in transformation. Further, site-specific mutagenesis was utilized to define regions of the 5' LTR required for A-MuLV transforming gene function in vivo. Removal of one or both 72 bp repeated units did not impair transforming ability. Deletion of either TATA or CAAT sequences, or sequences upstream from the 72 bp repeat, reduced but did not abolish transforming activity. Finally, deletions which encompassed TATA, CAAT and poly A signals completely abolished transformation.